Osteoprotegerin and RANKL in the pathogenesis of osteoporosis in patients with thalassaemia major.

Osteoporosis represents an important cause of morbidity in thalassaemia major patients; the etiopathogenesis is multifactorial and includes expansion of the bone marrow, endocrine disorders, iron overload and genetic factors. Two cytokines, osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL), have recently been identified as important mediators in the pathogenesis of osteoporosis. In this study, the possible role of the OPG-RANKL system in the pathogenesis of osteoporosis in thalassemia major is assessed, as well as any correlations between the serum levels of OPG and RANKL and bone mineral density (BMD), 17 beta-estradiol and free testosterone and the relationship between T-score of BMD and OPG/RANKL ratio. In 31 thalassaemia major patients and a control group, the serum values of OPG and RANKL were assayed and correlated with BMD, as well as with the sex hormones values. All the thalassemic patients had reduced BMD and 35.5% presented osteoporosis. The thalassemic patients had significantly higher serum levels of OPG than the controls, while their higher RANKL levels, were at the threshold of significance. The OPG/RANKL ratio showed higher level respect to the controls. No statistically significant correlation was observed between the T-score and RANKL neither between the T-score and OPG nor between T-score and OPG/RANKL ratio. Instead, a statistically significant correlation was found between the T-score and free testosterone and between the T-score and 17 beta-estradiol. There was no correlation between the sex hormones and OPG and RANKL. The increased OPG values in thalassemic patients could be considered to compensate the increased bone turnover. The authors confirm hypogonadism as a primary etiopathogenetic factor in the reduced BMD observed in thalassaemia major patients.

Non cardiopatic and cardiopatic beta thalassemic patients: quantitative and qualitative cardiac iron deposition evaluation with MRI.

PURPOSE: Cardiomyopathy is one of the major complications of b thalassaemia major as a result of transfusional iron overload. The aim of our study is to evaluate with MR if there is any difference of iron deposition signal intensity (SI) or distribution between non-cardiopathic and cardiopathic thalassaemic patients in order to establish if there is a relationship between cardiopathy and iron deposition. MATERIALS AND METHODS: We studied 20 patients affected by b thalassaemia major, of whom 10 cardiopathic and 10 non-cardiopathic, and 10 healthy volunteers as control group. Serum ferritin and left ventricular ejection fraction were calculated in thalassaemic patients. All patients were examined using a 1.5 MR unit with ECG-gated GE cine-MR T2*-weighted, SE T1-weighted and GE T2*-weighted sequences. In all cases, using an adequate ROI, the myocardial and skeletal muscle signal intensity (SI), the myocardial/skeletal muscle signal intensity ratio (SIR) and the SI average of the myocardium and skeletal muscle were calculated for every study group. The qualitative evaluation of iron deposition distribution was independently performed by three radiologists who analyzed the extension, the site and the morphology of iron deposition on the MR images and reported their observations on the basis of a four-level rating scale: 0 (absent), 1 (limited), 2 (partial), 3 (widespread deposition). The result of quantitative and qualitative evaluations were analysed with statistical tests. RESULTS: Cardiac iron deposition was found in 8/10 non-cardiopathic thalassaemic patients and in all cardiopathic thalassaemic patients. We noticed a significant SI difference (p>0.05) between the healthy volunteer control group and the thalassaemic patients with iron deposition, but no significant SI difference in iron deposition between non-cardiopathic and cardiopathic thalassaemic patients in the areas evaluated. The qualitative evaluation revealed a different distribution of iron deposition between the two thalassaemic groups, with more widespread distribution in cardiopathic patients. CONCLUSIONS: We found cardiac iron deposition also in non-cardiopathic b thalassaemic patients and a qualitative difference in cardiac iron distribution between non-cardiopathic and cardiopathic patients. The qualitative evaluation of cardiac iron deposition was useful for an easier classification of the disease, bypassing the SI quantitative value which is affected by the extremely uneven distribution of iron deposition and by the sampling technique used. MR evaluation of non-cardiopathic thalassaemic patients may be useful to evaluate early iron deposition and to establish the most suitable chelation therapy.

HFE gene mutations an Apulian population: allele frequencies.

Hereditary hemochromatosis (HH) is an autosomal recessive trait regarding iron metabolism frequently found in Caucasian populations. The C282Y mutation of the HFE gene, held responsible for HH, has been identified as the major genetic basis for the phenotypic expression of HH whereas two additional mutations of the HFE H63D and S65C gene appear to be associated with a milder form of HH. A high allele frequency of C282Y and H63D has been reported in Northern European populations. In Italy, the overall allele frequency was 0.5% for the C282Y mutation, 12.6% for the H63D mutation and 1.1% for the S65C mutation. In this study, we evaluated the allele frequency of the three principal HFE mutations (C282Y, H63D, S65C) together with eight additional mutations (V53M, H63H, Q127H, E168Q, E168stop, W169stop, V59M, Q238P) in 500 healthy Apulian subjects. No subject homozygous for the C282Y mutation was found while 3% of subjects were heterozygous for this mutation. Heterozygosity and homozygosity for the H63D mutation were 26 and 1%, respectively. Only five subjects were heterozygous for the S65C mutation. Overall, the allele frequency was 1.5% for C282Y, 14% for H63D, 0.5% for S65C and 0% for the other mutations. The transferrin saturation (TS) was significantly higher in subjects heterozygous for the H63D mutations with respect to subjects with a normal genotype, though all were within the normal range. No statistically significant difference in the allele frequency was noted in the Apulian population compared to that in Northern and Southern Italy.

Analysis of HLA-DRB1*-A* and -B* alleles in prenatal diagnosis for determination of maternal contamination in fetal DNA.

During chorionic villi sampling for prenatal diagnosis with molecular biology techniques, contamination by maternal decidua frequently occurs and can lead to misinterpretation of the test results. To avoid such problems, we present a new method for appraising maternal contamination of fetal DNA, based on genomic typing of the highly variable human leukocyte antigen (HLA) locus-DRB1*, locus A* and locus B* regions by genetic amplification with sequence-specific primers and PCR. Fetal DNA samples obtained for beta-thalassemia diagnosis were analysed after artificial contamination with increasing maternal DNA concentrations ranging from 0.5 to 10% (0.5, 1, 3, 5 and 10%). The approach was found to be rapid, specific, reproducible and highly sensitive and permits recognition of 1-3% contamination by maternal DNA concentrations. The system currently used for detecting maternal DNA contamination in fetal samples is the analysis of polymorphic loci by variable number of tandem repeats and/or short tandem repeats. We propose that the analysis of HLA alleles may provide a valid alternative or complement to this system.

Genotype and phenotype correlation in glucose-6-phosphate dehydrogenase deficiency.

BACKGROUND AND OBJECTIVES: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common erythrocytic enzymatic disorder in Italy and is characterized by wide clinical, biochemical and molecular variability. We studied the clinical and hematologic data from 54 G6PD-deficient, unrelated males from the Apulia region. DESIGN AND METHODS: Analyses for enzymatic activity, G6PD electrophoresis and molecular typing were performed on all subjects. Thirty-nine subjects (72.2%) showed a severe G6PD deficiency (<10% residual enzymatic activity) and 15 subjects (27.8%) a moderate deficiency (10--60% residual activity). RESULTS: The Mediterranean variant was found in 48.2% of cases, the Seattle variant in 33.3%, the A- variant in 7.45% and the Montalbano variant in 3.7%; the variant was not identified in four subjects. Thirty-two patients (59.2%) were asymptomatic; of these, 37.04% demonstrated acute hemolytic crises induced mainly by ingestion of fava beans and 3.7% had had neonatal jaundice. Acute hemolytic anemia was found in 53.8% of subjects with the Mediterranean variant, in 5.5% with the Seattle variant, in 100% with the A-variant and 0% with the Montalbano variant. INTERPRETATION AND CONCLUSIONS: Enzymatic activity was shown to be a poor predictive parameter of acute hemolytic crises and was not correlated with clinical features. Subjects with Mediterranean or A- variants had a more severe clinical phenotype which was not related to enzymatic activity. The Seattle, and probably the Montalbano, variant appears to have a milder clinical expression.

Lymphocyte subset reconstitution after HLA-identical placental blood transplantation (PBT) or PBT plus bone marrow transplantation (BMT) in three children with beta-thalassemia major.

The kinetics of circulating lymphoid cells were evaluated in three children suffering from beta-thalassemia major after HLA-identical sibling placental blood transplant (PBT) in one patient and placental blood plus bone marrow transplantation (BMT) in two patients. Recovery of the main lymphocyte subsets, as determined by phenotype analysis of circulating PBMCs, was complete within 2 months after transplant. NK (CD56+) cells were the first to appear in peripheral blood, followed by T (CD3+, CD2+, CD7+) and B (CD19+) cells. Of the T lymphocytes, the CD8+ were the first to reconstitute, but recovery of CD4+ cells was also rapid and within 6 months these T cells reached normal values. The expression of CD57 by NK or T cells was slightly delayed. The evaluation of RA and RO isoform expression of the CD45 molecule showed a prevalence of the CD45RA antigen with a ratio of 2-3:1. In the PBT only patient, T cells expressing the CD45RO antigen prevailed in the early post-transplant period. Severe or chronic GVHD was not observed. This experience demonstrates that reconstitution of lymphocyte subsets is successful in genetic hematological diseases after transplantation of HLA-identical placental blood or placental blood plus bone marrow from healthy or heterozygous siblings. Bone Marrow Transplantation (2000) 26, 743-747.

alpha-Thalassaemia in Apulia: biosynthetic studies.

Analysis of haemoglobin chain synthesis was performed in 15 Apulian patients with Hb H disease and in their patients and offspring. The Apulian carriers of Hb H disease show a marked imbalance of alpha and beta chain synthesis (0.39 +/- 0.1) with variable clinical and haematological manifestations. However, we are dealing with an intermediate form similar to that described in Italians from other regions. A significant difference was found between the mean alpha/beta ratio values (0.81 +/- 0.13) of parents and offspring of Hb H patients and those of the normal controls (1.05 +/- 0.09); however, extensive overlapping between these two groups exists. These results have led us to the conclusion that the forms of alpha-thalassaemia found in Apulia are similar to the alpha defects observed in Sicily; in both cases, in fact, haemoglobin chain synthesis was an unreliable test for discriminating between alpha-thalassaemia-1 trait and alpha-thalassaemia-2 trait.

Chronic viral hepatitis in thalassemic liver disease. A long-term study in patients with acute hepatitis with nontransfusion-dependent thalassemia minor.

To evaluate the effective role of hepatitis viruses in thalassemic (Th) liver disease, we carried out a long-term study in 42 subjects with nontransfusion-dependent Th minor hospitalized for an episode of acute viral hepatitis. 10 patients had serologic evidence of hepatitis A, 23 of hepatitis B and 9 of hepatitis non-A, non-B. In the follow-up chronic hepatitis was detected histologically in 5/23 patients with hepatitis B and 5/9 with hepatitis non-A, non-B. All hepatitis A patients recovered completely. The prevalence in 7 out of 10 patients with chronic hepatitis of piecemeal necrosis and of inflammatory changes over hepatic siderosis and fibrosis evidenced a determinant role of chronic viral infection in the development of liver damage in these patients. Thus, heterozygous nontransfusion-dependent Th patients seem to have a high risk of developing a chronic inflammatory liver disease especially after an episode of non-A, non-B hepatitis. Therefore, in our geographical area, chronic hepatitis of viral origin should be taken into account, among other pathogenetic factors, in many cases of cryptogenic thalassemic liver disease.